PROJECT SUMMARY/ABSTRACT Cancer cell proliferation requires a coordinated metabolic network that supports biomass accumulation and maintains cellular redox homeostasis. An essential mechanism that cancer cells rely on to achieve coordinated anabolic cell growth with redox balance is the inter-conversion of nicotinamide adenine dinucleotide phosphate between the oxidized (NADP+) and reduced (NADPH) forms (the total of NADP+ and NADPH is referred to as NADP(H) hereafter). Because NADP(H) molecules cannot cross membrane structures, cellular NADP(H) pools are compartmentalized. In the cytosol, NADPH acts as a cofactor for fatty acid biosynthesis, and as the reducing power for glutathione regeneration and anti-oxidant defense. Mitochondria are central organelles of cellular metabolism that participate in a number of biosynthetic pathways as well as redox regulation. However, the role of the mitochondrial NADP(H) pool in supporting cell proliferation remains to be elucidated. My preliminary results indicate that mitochondrial NADP(H) is not required for anti-oxidant defense as currently thought. Instead, its main function is to support de novo proline synthesis required to maintain cell proliferation. Cancer cell growth is stalled upon depletion of the mitochondrial NADP(H) pool. Supplementing exogenous proline is both necessary and sufficient to restore cell growth. The proposal is designed to further strengthen these observations by defining the mechanistic basis of how mitochondrial NADP(H) availability specifically determines proline synthesis, and to determine contexts in which targeting the mitochondrial NADP(H) pool is beneficial for cancer treatment. The following Specific Aims are pursued in this application: Aim 1. Investigate the function of the mitochondrial NADP(H) pool in supporting cancer cell growth (K99); Aim 2. Identify the molecular mechanisms whereby mitochondrial NADP(H) availability dictates proline biosynthesis (K99); Aim 3. Examine the vulnerability of cancer cells with isocitrate dehydrogenase 2 (IDH2) mutations by targeting mitochondrial NADP(H) (R00). The knowledge and scientific expertise that I acquire from these studies and throughout the award period will also facilitate my transition into the future independent career, with the long- term goal to study the mechanistic basis of cancer and develop clinical approaches for cancer treatment. In addition to the scientific goals, I have also outlined a detailed career development plan in this application in order to obtain skills that are important for leading an independent research laboratory. I will conduct the proposed research and carry out the training plan under the mentorship of Dr. Craig Thompson. I will embark on the academic environment provided by Memorial Sloan-Kettering Cancer Center to achieve these goals and transition to a position as an independent principle investigator of cancer biology.